Genetics and proteomics depend on the ability to analyze complex mixtures of biological origin with high sensitivity and maximum selectivity. Especially the rapid development of miniaturized techniques in analytical chemistry (He et al. Anal. Chem. 70:3790–3797 (1998)) has had a profound impact on the modern practice of analyzing biological samples of high complexity (Novotny J. Chromatogr. B 689: 55–70 (1997)). Several techniques based on the principle of differential migration (Rathore et al. J. Chromatogr. A 743: 231–246 (1996)) were developed after the introduction of fused silica capillaries to analytical chemistry (Dandeneau et al. HRC & CC: 2:351 (1979)), in particular capillary liquid chromatography (CLC) (Hirata et al. J. Chromatogr. 186:521–528 (1979)), capillary electrophoresis (CE) (Jorgenson et al. J. Chromatogr. 218:209–216 (1981)), and capillary electrochromatography (CEC) (Jorgenson et al. J. Chromatogr. 218:209–216 (1981)).
Columns packed with microparticulate sorbents have been successfully applied as separation media in high-performance liquid chromatography (HPLC). Despite many advantages, HPLC columns packed with microparticulate, porous stationary phases have some limitations, such as the relatively large void volume between the packed particles and the slow diffusional mass transfer of solutes into and out of the stagnant mobile phase present in the pores of the separation medium (Martin et a. Biochem J. 35:1358 (1941); Unger et al in Packings and Staionary Phases in Chromatographic Techniques, Unger Ed: Marcel Dekker: New York, p. 75 (1990)).
One approach to alleviate the problem of restricted mass transfer and intraparticular void volume is the concept of monolithic chromatographic beds, where the separation medium consists of a continuous rod of a rigid, polymer which has no interstitial volume but only internal porosity consisting of micropores and macropores. Monolithic separation columns are becoming more widely used in HPLC of biomolecules.
WO 97/19347 relates to a method and device for separating one or several organic substances in a sample. The chromatographic device comprises a monolith prepared in an emulsion system containing at least 75% by weight of water phase. Separations of polynucleotides were not disclosed.
U.S. Pat. No. 5,334,310 relates to a monolith containing small pores having diameters less than about 200 nm and large pores with diameters greater that about 600 nm. The columns were equipped with end fittings. No separations of polynucleotides were demonstrated.
WO 00/15778 relates to polymeric monolithic beds for resolving mixtures containing polynucleotides. However, single-stranded molecules were poorly resolved using the column. The columns had inner diameters (ID) of greater than 4 mm and were equipped retaining frits. The mobile phase buffers included EDTA. Useful separations of DNA fragments by IP-RP-HPLC using underivatized polystyrene/divinylbenzene monolithic columns could not be achieved and such columns were not recommended.
There is a need for improved monolithic columns and methods for the separation of polynucleotides.